Various diagnostic assays for the in vitro determination of haptens in body fluid are known, including specifically: radioimmunoassay (RIA), competitive protein-binding radioassay (CPB), enzyme immunoassay (EIA), enzyme multiplied immunological technique (EMIT), enzyme linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), and hemagglutination inhibition (HAI) assay. The assays are based on the principle of optimizing the mutual affinity between the hapten (sometimes referred to herein as compound, antigen, or ligand) and a specific antibody or other binding entity for that hapten. Inherent in these assays is the need for the physical joining of molecules of the hapten and binder. However, the haptens are frequently bound to specific binding proteins or lipoproteins, for example, thyroxine to thyroid binding protein and testosterone to testosterone binding protein. This hapten binding interferes with the intended hapten-binder joining. Also, the lipids present in body fluid such as blood plasma or serum ordinarily retard this joining, for example, by physical blocking of the two reactants or by combination or binding with either reactant. Prior attempts to overcome this problem have been unduly time-consuming and expensive. Radioimmunoassay and related methodologies (competitive protein binding radioassays, etc.) have traditionally been restricted to measurement of the "free" portion of a selected hapten when the hapten exists as a protein or lipoprotein complex. Only that percentage of the total hapten concentration in any body fluid which existed free of protein or lipoprotein association was available to join with the exogenous binder employed for purposes of the assay. The only remedy prior to this invention to accomplish a total hapten measurement was to disassociate the hapten-protein bond by means of an organic extraction or other laborious procedure (ultracentrifugation, dialysis, etc.).
The incorporation of surfactant as will be described subsequently serves to break the endogenous hapten binding and make the total concentration available to join with another binding entity unaffected by the presence of surfactants. This surfactant may be combined with the exogenous binder to allow a direct assay of total hapten, completely free of extra manipulative procedures. These attempts have included, for example, ultracentrifugation, microfiltration, alkaline hydrolysis and extraction with organic solvents to remove lipids, and selective dialysis and extraction to isolate the non-lipid fraction. Despite these attempts, the art has lacked an effective method, and especially a cost-effective method, of assaying lipid-containing body fluid.
It is therefore an object of the present invention to provide cost-effective means for assaying the content of selected protein or lipoprotein bound hapten (compound, antigen or ligand), by direct assay without prior extraction or pretreatment.
A further object of the invention is to provide broadly applicable means for immunoassay of any of various exogenous or endogenous haptens, compounds, antigens or ligands in lipid-containing body fluid such as serum or plasma.
These and other objects, features and advantages of the present invention will become apparent from the following description.